The present invention relates generally to choline binding polypeptides and to nucleic acids encoding such polypeptides. The invention also relates to vaccines which provide protection or elicit protective antibodies to bacterial infection, and to antibodies and antagonists against or inhibitors of such polypeptides for use in diagnosis, therapy and passive immune therapy. In particular, the choline binding polypeptides of the invention are useful as vaccines against Streptococcus, particularly pneumococcus. A choline binding polypeptide of the present invention is also useful as a competitive inhibitor of bacterial adhesion, or to discover small molecule antagonists of adhesion.
Streptococcus pneumoniae is a gram positive bacteria which is a major cause of invasive infections such as sepsis, meningitis, otitis media and lobar pneumonia (Tuomanen et al NEJM 322:1280-1284, 1995). Vaccination has long been an important armament in the arsenal against infectious microorganisms. Prior to the introduction of antibiotics, vaccination was the major hope for protecting populations against viral or bacterial infection. With the advent of antibiotics, vaccination against bacterial infections became less important. However, the emerging problems withantibiotic resistance among such infectious bacteria, including S. pneumoniae strains, have raised an urgent need for a better understanding of the pathogenesis of these pathogens and has reestablished the importance of anti-bacterial vaccines (Appelbaum, P.C. (1992) Clin Infect Dis 15:77-83).
There are over 90 different types of the pneumococcal organism, each with a different chemical structure of the capsular polysaccharide (i.e., each antigenically distinct). The capsular polysaccharide is a principal known virulence factor of the pneumococcus and induces an antibody response in adults. Current vaccines against S. pneumoniae employ mixtures of the capsules of the 23 most common serotypes of this bacterium. After several comprehensive studies there is now overwhelming evidence that this vaccine is approximately 60% efficacious for the general population (Shapiro et al.(1991) NEJM 325:1453-60). Importantly, these vaccines are ineffective in individuals most susceptible to pathological infectionxe2x80x94the young, the old, and the immune compromisedxe2x80x94because of their inability to elicit a T cell immune response. Conjugation of the capsule to a protein permits protection in the younger age group but is inherently limited in the number of capsules that can be conjugated at one time (approximately 5-8 capsules only).
Exported proteins in bacteria participate in many diverse and essential cell functions such as motility, signal transduction, macromolecular transport and assembly, and the acquisition of essential nutrients. For pathogenic bacteria such as S. pneumoniae, many exported proteins are virulence determinants that function as adhesins to colonize and thus infect the host, or as toxins to protect the bacteria against the host""s immune system (for a review, see Hoepelman and Tuomanen (1992) Infect Immun 60:1729-33). One alternative to current vaccines are subunit vaccines in which the antigen, or antigens, include a bacterial surface protein or proteins. These vaccines could overcome the deficiencies of whole bacterial or capsule-based vaccines. Moreover, given the importance of exported or surface proteins to bacterial virulence, these proteins are an important target for therapeutic intervention.
Pneumococci bind avidly to cells of the upper and lower respiratory tract. Like most bacteria, adherence of pneumococci to human cells is achieved by presentation of bacterial surface proteins that bind to eukaryotic cell surface carbohydrates in a lectin-like fashion (Cundell, D. and Tuomanen, E. (1994) Microb Pathog 17:361-374). Pneumococci bind to non-inflamed epithelium, a process that can be viewed as asymptomatic carriage. It has been proposed that the conversion to invasive disease involves the local generation of inflammatory factors which, activating the human epithelial cell, change the number and type of receptors available on the human cells (Cundell, D. et al. (1995) Nature, 377:435-438). Presented with an opportunity in this new setting, pneumococci appear to take advantage and engage one of these unregulated receptors, the platelet activating factor (PAF) receptor (Cundell et al. (1995) Nature, 377:435-438). Within minutes of the appearance of the PAF receptor, pneumococci undergo waves of enhanced adherence and invasion. Inhibition of bacterial binding to activated cells, for instance by soluble receptor analogs, blocks the progression to disease in animal models (Idanpaan-Heikkila, I. et al. (1997) J. Infect. Dis., 176:704-712). Particularly effective in this regard are soluble carbohydrates containing lacto-N-neotetraose with or without an additional sialic acid, which prevent pneumococcal attachment to human cells in vitro and prevent colonization in the lung in vivo.
Pneumococci display a family of surface proteins which bind to the bacterial surface by non-covalent association to the cell wall teichoic acid or lipoteichoic acid, specifically through its terminal component phosphorylcholine. The surface of Streptococcus pneumoniae is decorated with twelve types of these choline binding proteins (Cbps). The Cbps are represented by a family of molecules which decorate the surface of pneumococcus, each serving a unique function but bound to the surface by a common element. These proteins consist of an N-terminal activity domain and a repeated C-terminal signature choline binding domain that contains two to greater than ten repeats of a 20 amino acid choline binding sequence that binds to phosphoryl choline and that anchors these molecules to the surface of the bacteria. This motif has been identified in the C-terminal regions of a secreted glycoprotein from Clostridium acetobutylicum NCIB 88052 [Sanchez-Beato, et al., J. Bacteriol. 177:1098-1103 (1995)], toxins A and B from Clostridium difficile [Von Eichel-Streiber and Sauerbom, Gene 96:107-13 (1990); Von Eichel-Streiber et al., J. Bacteriol. 174:6707-6710 (1992)], a glucan-binding protein from Streptococcus mutans, several glycosyltransferases from Streptococcus downei and S. mutans, the murein hydrolase (LytA) from pneumococcus and pneumococcal lytic phage [Ronda et al., Eur. J. Biochem. 164:621-4 (1987); Diaz et al., J. Bacteriol. 174:5516-25 (1992); Romero et al., Microb. Lett. 108:87-92 (1993); Yother and White, J. Bacteriol. 176:2976-85 (1994)], and a surface antigen (PspA) also from pneumococcus. The known pneumococcal Cbp family members are CbpA, LytA and PspA.
The choline binding domain was recognized and fully characterized by Lopez et al. in his studies of the autolytic enzyme, LytA (Ronda et al. (1987) Eur. J. Biochem, 164:621-624). From studies of the sequences of the recognized choline binding proteins, consensus sequences have been reported, most particularly that of Garcia et al: GWLKDNGSWYYLNANGAMAT (SEQ ID NO:26) (Garcia, P. et al (1990) Gene 86:81-88; Wren B. et al (1991)Mol Microbiol 5:797-803); Sanchez-Beato, A. R. et al (1995) J Bacteriol 177:1098-1103).
Teichoic acid (TA), an integral part of the cell wall of Streptococcus pneumoniae, contains many terminal phosphorylcholine moieties. Choline affinity chromatography or Mono-Q Sepharose, a close relative of choline, were used to purify the CBPs. Previous studies have shown that PspA, as well as one other surface exposed protein, LytA, the autolytic amidase, bind in a choline-dependent manner. PspA, a protein having a molecular weight of 84 kDa, and which is highly variable, is released from the cell surface with high choline concentration (at least about 2% to about 10%). The function of PspA is unknown. LytA, or autolysin, is a 36 kDa protein, which lyses the pneumococcal cell wall (self lysis), but is not released from the cell by growth in high concentrations of choline, by washing in 10% choline, or by growth in ethanolamine. Reports on choline binding proteins include those by Sanchez-Puelles et al Gene 89:69-75 (1990), Briese and Hakenback Eur. J. Biochem. 146:417-427, Yother and White J. of Bacteriol. 176:2976-2985, Sanchez-Beato et al J. of Bacteriol. 177:1098-1103, and Wren Micro. Review Mol. Microbiol. 5:797-803 (1991), which are hereby incorporated by reference in their entirety.
CbpA is an adhesin (ligand) for the glycoconjugate containing receptors present on the surface of eucaryotic cells. CbpA is a 663 amino acid protein with an apparent molecular mass of 112 KDa. CbpA has been shown to be critical to pneumococcal colonization and attachment to human cells. Mutants with defects in cbpA show reduced virulence in the infant rat model for nasopharyngeal colonization and fail to bind to eucaryotic cells found at the site of infection and to glycoconjugates that bind pneumococcus. The CbpA protein cross reacts with human convalescent antisera, and antisera to the Cbps passively protected mice in a model for sepsis. Since the process of colonization and the progression to disease depend on pneumococcal attachment to human cells as a primary step, interruption of the function of surface choline binding proteins or the choline binding domain, for instance by cross reactive antibody or by inhibition with a competitive peptide mimicking this domain, may be relevant to blocking disease.
Choline-binding proteins for anti-pneumococcal vaccines are disclosed in International Patent Application WO 97/41151 of Masure, et al, which claims priority to U.S. patent application Ser. No. 08/642,250, which are hereby incorporated by reference in their entirety. The International patent Application WO 97/41151 discloses partial polypeptide sequences of certain putative choline binding proteins and the polypeptide sequence of CbpA.
The cell wall associated choline binding protein, LytA, a murein amidase, is an autolysin that is responsible for remodeling the cell wall, the separation of daughter cells, cell death in stationary phase and penicillin induced cell lysis. Expansion of the cell wall during bacterial growth and splitting of the septum for cell separation require enzymes that cleave the peptidoglycan network enclosing the cell. In addition to acting as spacemaker enzymes for cell wall growth, some of these enzymes act as autolysins, thereby representing potentially suicidal enzymes. Regulation of these enzymes is therefore important and must take into consideration their extracytoplasmic location. Unlike other bacteria which have multiple autolysins, S. pneumoniae has only one major autolysin, LytA. Antibiotics such as penicillin induce bacteriolysis by interfering with the control of the endogenous autolysins (Tomasz and Holtje, 1977, in Microbiology, D. Schlessinger, ed., pp. 202-215; Tomasz, 1983, in The Target of Penicillin, R. Hackenback et al eds., pp. 155-172). Although the binding of antibiotics to cell wall synthetic enzymes has been well characterized, the mechanism by which it induces autolysin mediated cell wall degradation is unknown. Tolerance to such antibiotics arises if the bacterial autolysins are not triggered as the antibiotic inhibits the cell wall synthetic machinery. The question of whether antibodies raised against LytA are protective against infection by S. pneumoniae and its requirement for virulence remains controversial. In some cases, loss of LytA by deletion leads to less virulence, while others report no effect (Berry, A. M. et al (1992) Microb Pathog 12:87-93).
PspA has been reported to be a candidate for a S. pneumoniae vaccine as it has been found in all pneumococci to date [see Yother et al., J. Bacteriol., 174:610 (1992)]; the purified protein can be used to elicit protective immunity in mice; and antibodies against the protein confer passive immunity in mice [Talkington et al., Microb. Pathog. 13:343-355 (1992)]. However, PspA demonstrates antigenic variability between strains in the N-terminal half of the protein, which contains the immunogenic and protection eliciting epitopes (Yother et al., supra). This protein does not represent a common antigen for all strains of S. pneumoniae, and therefore is not an optimal vaccine candidate.
As described above, each Cbp, while being bound to the surface by a common element, the choline binding domain, serves a unique function. This particular function is largely determined by the unique N-terminal activity domain of each Cbp. N-terminal domain fragments of Cpbs, particularly CbpA, have been shown to have activity in blocking adherence and are candidates for immunogenic vaccines, independent of the C-terminal choline binding domain. In particular, Tuomanen et al, U.S. Ser. No. 09/056,019, which is hereby incorporated herein by reference in its entirety, provides isolated polypeptides comprising N-terminal choline binding protein A truncates, particularly wherein the polypeptides do not bind to choline. Vaccines comprising such N-terminal truncates, DNA encoding such truncates, or antibodies directed against such truncates are also described in U.S. Ser. No. 09/056,019.
An additional surface exported protein that affects adherence and represents a virulence determinant for pneumococcus, ZmpB, has been identified and described by R. Novak and R. Masure in U.S. Ser. No. 09/096,336 filed Jun. 11, 1998 which is hereby incorporated by reference in its entirety. ZmpB is a zinc metalloprotease, dependent on Zn2+ for functional activity. zmpB mutants show loss of adherence and colonization and are defective in the autolytic pathway. A mutation in zmpB selectively alters the production of CbpA and generates covalently modified LytA. ZmpB is proposed to be a master regulatory protein that controls the expression of multiple Cbps critical for bacterial survival in the human host. ZmpB is a candidate for an S. pneumoniae vaccine or as one component of a multi-component vaccine.
The deficiencies of whole bacterial or capsule-based vaccines and the emerging problems with antibiotic resistance among infectious bacteria, including S. pneumoniae strains, have raised an urgent need for alternative effective vaccines and therapies. The invention herein fills such a need in providing protective vaccines and therapies.
The citation of references herein shall not be construed as an admission that such is prior art to the present invention.
In its broadest aspect, the present invention encompasses isolated polypeptides comprising an amino acid sequence of a choline binding protein, CbpG.
The present invention is directed to an isolated polypeptide comprising an amino acid sequence of a pneumococcal choline binding protein, CbpG and wherein such polypeptide contains a choline binding domain sequence which is homologous to SEQ ID NO:1.
The present invention provides an isolated polypeptide comprising an amino acid sequence of a pneumococcal choline binding protein, CbpG wherein such polypeptide contains a choline binding domain sequence which is homologous to GWLKDNGSWYYLNANGAMAT (SEQ ID NO:10).
The present invention further provides an isolated polypeptide comprising an amino acid sequence of an N-terminal CbpG choline binding protein truncate. The present invention provides an N-terminal CbpG truncate comprising the amino acid sequence as set forth in SEQ ID NO: 11. The invention particularly provides an N-terminal CbpG truncate consisting of amino acids 1-90 of the CbpG choline binding protein. In a further embodiment, the N-terminal CbpG truncate comprises less than amino acids 1-90 of the CbpG choline binding protein.
In a still further aspect, the present invention extends to vaccines based on the choline binding proteins described herein.
The present invention provides an isolated polypeptide comprising an amino acid sequence of a choline binding protein CbpG. The polypeptide comprises the amino acid sequence as set forth in SEQ ID NO:2 including fragments, mutants, variants, analogs, or derivatives, thereof The isolated polypeptide is suitable for use in immunizing animals and humans against bacterial infection, preferably pneumococci.
The present invention further provides an isolated polypeptide comprising an amino acid sequence of a N-terminal CbpG choline binding protein truncate, particularly wherein the polypeptide does not bind to choline. This invention provides an isolated immunogenic polypeptide comprising an amino acid sequence comprising an N-terminal truncate of a choline binding protein CbpG. In a particular embodiment, this invention provides an isolated immunogenic N-terminal CbpG truncate polypeptide comprising the amino acid sequence as set forth in SEQ ID NO:11. The present invention further particularly provides an immunogenic N-terminal CbpG truncate polypeptide consisting of amino acids 1-90 of CbpG, or of less than amino acid 1-90 of CbpG.
In a still further aspect, the present invention extends to an immunogenic choline binding protein polypeptide CbpG or a fragment thereof The present invention also extends to immunogenic choline binding protein polypeptides wherein such polypeptides comprise a combination of at least two choline binding polypeptides selected from the group consisting of CbpG and at least one other choline binding polypeptide, including fragments thereof and N-terminal truncates thereof.
The present invention also relates to isolated nucleic acids, such as recombinant DNA molecules or cloned genes, or degenerate variants thereof, mutants, analogs, or fragments thereof, which encode the isolated polypeptide or which competitively inhibit the activity of the polypeptide. The present invention further relates to isolated nucleic acids, such as recombinant DNA molecules or cloned genes, or degenerate variants thereof, mutants, analogs, or fragments thereof, which encode the choline binding protein CbpG. Preferably, the isolated nucleic acid, which includes degenerates, variants, mutants, analogs, or fragments thereof, has a sequence as set forth in SEQ ID NO:3. In a further embodiment of the invention, the full DNA sequence of the recombinant DNA molecule or cloned gene so determined may be operatively linked to an expression control sequence which may be introduced into an appropriate host. The invention accordingly extends to unicellular hosts transformed with the cloned gene or recombinant DNA molecule comprising a DNA sequence encoding the present invention, and more particularly, the DNA sequences or fragments thereof determined from the sequences set forth above.
In a particular embodiment, the nucleic acid has the sequence selected from the group comprising SEQ ID NO:3; a sequence complementary to SEQ ID NO:3; or a homologous sequence which is substantially similar to SEQ ID NO:3. In a further embodiment, the nucleic acid has the sequence consisting of SEQ ID NO:3.
The present invention further relates to isolated nucleic acids encoding an N-terminal choline binding protein truncate, particularly wherein the encoded polypeptide does not bind to choline. This invention particularly provides isolated nucleic acids encoding N-terminal choline binding protein truncates of choline binding protein CbpG. The invention further provides a nucleic acid encoding an N-terminal CbpG truncate comprising SEQ ID NO:12, a sequence complimentary to SEQ ID NO:12, or a homologous sequence which is substantially similar to SEQ ID NO:12. In a further embodiment, the nucleic acid encoding the N-terminal CbpG truncate has the sequence consisting of SEQ ID NO:12.
In a particular embodiment, the nucleic acid encoding an N-terminal CbpG truncate is capable of encoding amino acids 1-90 of CbpG. In a still further embodiment, the nucleic acid encoding an N-terminal CbpG truncate encodes a truncate smaller than amino acids 1-90 of CbpG.
Nucleic acid vaccines or DNA vaccines utilize nucleic acids encoding particular immunogenic polypeptides to induce immunity in a host against such encoded immunogenic vaccines. Such nucleic acid based vaccines can be used directly as naked DNA, or can utilize well recognized expression vectors or retroviral vectors, as more particularly described herein, to encode such immunogenic polypeptide on expression in the host cell. Methods to generate and utilize such any such nucleic acid vaccines or DNA vaccines are well known in the art.
The present invention relates to nucleic acid vaccines or DNA vaccines comprising nucleic acids encoding immunogenic polypeptides of a choline binding protein CbpG. The present invention relates to nucleic acid vaccines or DNA vaccines comprising nucleic acids encoding immunogenic polypeptides of choline binding protein CbpG or a fragment thereof or any combination of CbpG with at least one other choline binding polypeptide. Still further, this invention provides nucleic acid vaccines or DNA vaccines comprising nucleic acids encoding an immunogenic N-terminal polypeptide of choline binding protein CbpG.
Antibodies against the isolated polypeptide include naturally raised and recombinantly prepared antibodies. These may include both polyclonal and monoclonal antibodies prepared by known genetic techniques, as well as bi-specific (chimeric) antibodies, and antibodies including other functionalities suiting them for diagnostic use. Such antibodies can be used in immunoassays to diagnose infection with a particular strain or species of bacteria. The antibodies can also be used for passive immunization to treat an infection with Gram positive bacteria, particularly pneumococcus. These antibodies may also be suitable for modulating bacterial adherence including but not limited to acting as competitive agents.
It is still a further object of the present invention to provide a method for the treatment of mammals to control the amount or activity of the pneumococci bacteria or its subunits, so as to treat or avert the adverse consequences of invasive, spontaneous, or idiopathic pathological states. This invention provides pharmaceutical compositions for use in therapeutic methods which comprise or are based upon the isolated polypeptides, their subunits or their binding partners.
The invention further provides pharmaceutical compositions, vaccines, and diagnostic and therapeutic methods of use thereof
The invention provides pharmaceutical compositions comprising a choline binding polypeptide CbpG and a pharmaceutically acceptable carrier. The present invention further provides pharmaceutical compositions comprising a choline binding polypeptide of a choline binding protein CbpG, or a fragment thereof or any combination of CbpG with at least one other choline binding polypeptide, and a pharmaceutically acceptable carrier. Still further, this invention provides pharmaceutical compositions comprising N-terminal polypeptide of choline binding protein CbpG and a pharmaceutically acceptable carrier.
The invention further relates to a vaccine for protection of an animal subject from infection with a Gram positive bacterium comprising a vector containing a gene encoding a choline binding protein CbpG of a Gram positive bacterium operatively associated with a promoter capable of directing expression of the gene in the subject. Preferably, such vaccine contains a gene encoding a choline binding protein CbpG of pneumococcus.
In another aspect, the invention is directed to a vaccine for protection of an animal subject from infection with a Gram positive bacterium, most preferably pneumococcus, comprising an immunogenic amount of a choline binding protein CbpG, or a derivative or fragment thereof. Such a vaccine may contain the protein conjugated covalently to a bacterial capsule or capsules from one or more strains of bacteria, including pneumococcus. In one such embodiment, at least one of the bacterial capsules is derived from a mutant strain of bacteria which is non-adherent and non-virulent. In a further such embodiment, the non-adherent and non-virulent bacteria is a pneumococcus and is a CbpG mutant bacteria. Such non-adherent and non-virulent CbpG mutant bacteria can further be utilized in expressing other immunogenic or therapeutic proteins for the purposes of eliciting immune responses to any such other proteins in the context of vaccines and in other forms of therapy.
The invention further provides a cbpG mutant bacteria which is non-adherent to nasopharyngeal cells or to lung cells. Particularly, such cbpG mutant is a gram positive bacteria. More particularly, such cbpG mutant is Streptococcus. Most particularly, such cbpG mutant is Streptococcus pneumoniae. 
The invention includes an assay system for screening of potential compounds effective to modulate the choline binding activity of the choline binding protein of the present invention. The invention more particularly includes an assay system for screening of potential compounds effective to modulate the choline binding activity of the choline binding protein CbpG. In one instance, the test compound, or an extract containing the compound, could be administered to a cellular sample expressing the choline binding protein CbpG to determine the compound""s effect upon the activity of the protein by comparison with a control. In a further instance the test compound, or an extract containing the compound, could be administered to a cellular sample expressing the choline binding protein CbpG, to determine the compound""s effect upon the activity of the protein, and thereby on adherence of said cellular sample to host cells, by comparison with a control.
The invention includes an assay system for screening of potential compounds effective to modulate the activity of the N-terminal domain of the choline binding protein of the present invention. The invention more particularly includes an assay system for screening of potential compounds effective to modulate the activity of the N-terminal domain of CbpG. In one instance, the test compound, or an extract containing the compound, could be administered to a cellular sample expressing the choline binding protein CbpG to determine the compound""s effect upon the activity of the protein by comparison with a control. In a further instance the test compound, or an extract containing the compound, could be administered to a cellular sample expressing the choline binding protein CbpG to determine the compound""s effect upon the activity of the protein, and thereby on adherence of said cellular sample to host cells, by comparison with a control. In another instance, the test compound, or an extract containing the compound, could be administered to a cellular sample expressing an N-terminal truncate (which lacks the C-terminal choline binding domain) of the choline binding protein CbpG to determine the compound""s effect upon the activity of the protein by comparison with a control. In a particular such instance, the cellular sample expresses an N-terminal CbpG truncate comprising amino acids 1-90 of CbpG or a smaller N-terminal truncate comprising less than amino acids 1-90 of CbpG. In a further such instance, the N-terminal CbpG truncate comprises the amino acid sequence set out in SEQ ID NO:11.
Other objects and advantages will become apparent to those skilled in the art from a review of the following description which proceeds with reference to the following illustrative drawings.